DESCRIPTION (Applicant's Abstract): A rapid, sensitive and widely applicable biochemical genomics approach has recently been developed to identify genes from the yeast Saccharomyces cerevisiae that specify biochemical activities. To this end, an available genomic set of ORFs (open reading frames) was used to construct an array of 6144 individual yeast strains, each expressing a different yeast open reading frame (ORFs) fused at its N-terminus to glutathione S-transferase (GST). To identify ORF-associated activities, strains were grown in defined pools and GST-ORFs were purified; then pools were assayed for activities, and active pools were deconvoluted to identify the source strain and GST-ORF associated with activity. In this way 14 different activities have been linked to a specific GST-ORF, including five activities that modify proteins or process RNA, four activities that can act on small molecules, and five activities that bind DNA or modulate DNA binding of other proteins. In principle this biochemical genomics approach can be used to identify the GST-ORF associated with any detectable activity, provided that it is functional, solubilized during extraction, and purifies with other required components. This approach is rapid; starting with the pools of purified GST-ORFs, it takes about two weeks to identify an ORF-associated activity. It is also sensitive because the purified GST-ORF pools can be assayed for hours. The goal of this proposal is to enhance the repertoire of this biochemical genomics approach in two ways: First, the number of biochemically functional ORF fusions will be expanded by making a C-terminal ORF-fusion library (since a large number of ORFs are not functional as N-terminal fusions, including many membrane proteins), and by adding several hundred ORFs currently not in the library. With these ORF-fusion strains, virtually every gene in yeast will be amenable to this biochemical genomics approach. Second, this approach will be extended to membrane-associated proteins, which comprise as many as 30 percent of the proteins in yeast, and are historically more difficult to purify. Using a variety of known activities, we will develop methods to purify and assay pools of membrane-associated ORF-fusions. Then we will apply these methods to two activities, which have not previously been linked to ORFs: (1) an enzyme catalyzing the attachment of palmitate to proteins, and (2) a protease responsible for degradation of the yeast mating pheromone a-factor. Application of these techniques to other organisms, including humans and pathogens, will greatly accelerate biochemical analysis and can be used to rapidly identify drug targets.